Teresa Neuparth

Effects of Temperature and Salinity on Life History of the Marine Amphipod Gammarus locusta. Implications for Ecotoxicological Testing

The life history of Gammarus locusta was analysed in the laboratory under the following temperature and salinity combinations: 20 °C–33‰, 15 °C–20‰ and 15 °C–33‰ (reference condition). Life history analysis comprised survival, individual growth, reproductive traits and life table parameters. Compared to 15 °C, life history at 20 °C was characterised by at least a four-week reduction in the life-span, lower life expectancy, shorter generation time, faster individual growth, anticipation of age at maturity and higher population growth rate. These temperature effects constituted an acceleration and condensation of the life cycle, compared to the reference condition. Concerning salinity effects, with few exceptions, results show that overall this amphipod life history did not differ significantly between the salinity conditions tested. Regarding ecotoxicological testing implications, findings from this study indicate that the range of temperature and salinity conditions acceptable for testing was substantially expanded both for acute and chronic assays. A temperature of 20 °C or higher (for a salinity of 33‰) is suggested for routine chronic sediment toxicity testing with G. locusta, in order to reduce the life cycle and consequently improve cost-effectiveness and standardisation. Results also suggest that a multiple-response approach, including survival, growth and reproduction, should be applied in chronic toxicity tests.

Assessment of the genotoxic potential of contaminated estuarine sediments in fish peripheral blood: laboratory versus in situ studies.

Juvenile Senegalese soles (Solea senegalensis) were exposed to estuarine sediments through 28-day laboratory and in situ (field) bioassays. The sediments, collected from three distinct sites (a reference plus two contaminated) of the Sado Estuary (W Portugal) were characterized for total organic matter, redox potential, fine fraction and for the levels of metals, polycyclic aromatic hydrocarbons (PAHs) and organochlorines, namely polychlorinated biphenyls (PCBs) and dichloro diphenyl tricholoethane plus its main metabolites (DDTs). Genotoxicity was determined in whole peripheral blood by the single-cell gel electrophoresis (SCGE or comet) assay and by scoring erythrocytic nuclear abnormalities (ENA). Analysis was complemented with the determination of lipid peroxidation in blood plasma by the thiobarbituric acid reactive substances (TBARS) protocol and cell type sorting. The results showed that exposure to contaminated sediments induced DNA fragmentation and clastogenesis. Still, laboratory exposure to the most contaminated sediment revealed a possible antagonistic effect between metallic and organic contaminants that might have been enhanced by increased bioavailability. The laboratory assay caused a more pronounced increase in ENA whereas a very significant increase in DNA fragmentation was observed in field-tested fish exposed to the reference sediment, which is likely linked to increased lipid peroxidation that probably occurred due to impaired access to food. Influence of natural pathogens was ruled out by unaltered leukocyte counts. The statistical integration of data correlated lipid peroxidation with biological variables such as fish length and weight, whereas the genotoxicity biomarkers were more correlated to sediment contamination. It was demonstrated that laboratory and field bioassays for the risk assessment of sediment contamination may yield different genotoxicity profiles although both provided results that are in overall accordance with sediment contamination levels. While field assays may provide more ecologically relevant data, the multiple environmental variables may produce sufficient background noise to mask the true effects of contamination.

Hazardous and Noxious Substances (HNS) in the marine environment: Prioritizing HNS that pose major risk in a European context

Increases in the maritime transportation of Hazardous and Noxious Substances (HNS), alongside the need for an effective response to HNS spills have led environmental managers and the scientific community to focus attention on HNS spill preparedness and responsiveness. In the context of the ARCOPOL project, a weight-of-evidence approach was developed aimed at prioritizing HNS that pose major environmental risks to European waters. This approach takes into consideration the occurrence probability of HNS spills in European Atlantic waters and the severity of exposure associated with their physico-chemical properties and toxicity to marine organisms. Additionally, a screening analysis of the toxicological information available for the prioritization of HNS was performed. Here we discuss the need for a prioritization methodology to select HNS that are likely to cause severe marine environmental effects as an essential step towards the establishment of a more effective preparedness and response to HNS incidents.

Hypocholesterolaemic pharmaceutical simvastatin disrupts reproduction and population growth of the amphipod Gammarus locusta at the ng/L range

Simvastatin (SIM), a hypocholesterolaemic drug, is among the most widely used pharmaceuticals worldwide and is therefore of emerging environmental concern. Despite the ubiquitous nature of SIM in the aquatic ecosystems, significant uncertainties exist about sublethal effects of the drug in aquatic organisms. Therefore, here we aimed at investigating a multi-level biological response in the model amphipod Gammarus locusta, following chronic exposures to low levels of SIM (64 ng/L to 8 μg/L). The work integrated a battery of key endpoints at individual-level (survival, growth and reproduction) with histopathological biomarkers in hepatopancreas and gonads. Additionally, an individual-based population modelling was used to project the ecological costs associated with long-term exposure to SIM at the population level. SIM severely impacted growth, reproduction and gonad maturation of G. locusta, concomitantly to changes at the histological level. Among all analysed endpoints, reproduction was particularly sensitive to SIM with significant impact at 320 ng/L. These findings have important implications for environmental risk assessment and disclose new concerns about the effects of SIM in aquatic ecosystems.

Review of oil and HNS accidental spills in Europe: identifying major environmental monitoring gaps and drawing priorities.

The European Atlantic area has been the scene of a number of extensive shipping incidents with immediate and potential long-term impacts to marine ecosystems. The occurrence of accidental spills at sea requires an effective response that must include a well executed monitoring programme to assess the environmental contamination and damage of the affected marine habitats. Despite a number of conventions and protocols developed by international and national authorities that focused on the preparedness and response to oil and HNS spills, much remains to be done, particularly in relation to the effectiveness of the environmental monitoring programmes implemented after oil and HNS spills. Hence, the present study reviews the status of the environmental monitoring programmes established following the major spill incidents over the last years in European waters, aiming at identifying the key monitoring gaps and drawing priorities for an effective environmental monitoring of accidental spills.

Toxicity ranking of estuarine sediments on the basis of Sparus aurata biomarkers.

Sparus aurata biomarkers were used to rank sediments from the Sado River estuary (Portugal) according to their toxicity. Initially, the activities of liver ethoxyresorufin-O-deethylase, liver and gill glutathione S-transferases, muscle lactate dehydrogenase, and brain acetylcholinesterase were tested in a laboratory bioassay with the reference compound benzo[a]pyrene. Enzymatic activities were determined in different tissues of fish exposed for 48, 96, or 240 h to three concentrations of benzo[a]pyrene (25, 50, and 100 microg/L). Induction of liver ethoxyresorufin-O-deethylase was observed at all the exposure periods and concentrations, suggesting a continuous response of this system to toxicant exposure. Induction of liver glutathione S-transferases activity was only observed after 240 h of exposure, whereas gill glutathione S-transferases activity was significantly inhibited at all the exposure periods, suggesting a direct or indirect effect of the toxicant on these enzymes. Inhibition of lactate dehydrogenases activity was only observed after 96 h of exposure to 25 microg/L of benzo[a]pyrene. No significant effects were observed on acetylcholinesterase activity, suggesting that cholinergic function of S. aurata is not affected by benzo[a]pyrene. In a second phase, fish were exposed for 240 h to sediments collected at five sites of the Sado River estuary, and the same biomarkers were analyzed. For all the enzymes assayed, significant differences among sites were found. In this study, the battery of biomarkers used allowed to discrimination among sites with different types of contamination, levels of contamination, or both, after multivariate data analysis. Discrimination of sites was similar to the ranking provided by a more complex and parallel study (including chemical analysis of sediments, macrobenthic community analysis, amphipod mortality toxicity tests, and sea urchin abnormality embryo assays), suggesting its suitability to evaluate the toxicity of estuarine sediments.

Detection of DNA strand breakage in a marine amphipod by agarose gel electrophoresis: exposure to X-rays and copper

This article describes the leading steps to develop an assay of DNA damage for the marine amphipod Gammarus locusta, using agarose gel electrophoresis (AGE). To test the sensitivity and feasibility of the AGE technique, X-ray assays were performed with naked DNA and with live amphipods. These positive controls demonstrated the effectiveness of the AGE technique to not only discriminate distinct levels of DNA strand breakage in a dose-dependent manner, but also to identify and quantify the type of strand breakage induced. It was also shown that it is possible to detect DNA damage using whole-body DNA extracts from amphipods. To explore the potential of this technique for use in ecotoxicological studies with amphipods, a 96-h waterborne-copper toxicity test was performed. Copper-induced DNA strand breakage was first observed after 24 h of exposure, and was recorded again at 96 h, at a copper concentration of 20 μg l -1 . The absence of strand breakage after 48 h of exposure is discussed in the light of the underlying mechanisms of copper toxicity and DNA repair. These studies demonstrated the feasibility of including DNA damage as a biomarker in ecotoxicological studies with amphipods. Information gained from the use of this biomarker would help with the interpretation of chronic toxicity tests and would contribute to our understanding of the impact of genotoxic insult in marine invertebrates, particularly crustaceans.

Simulation of a Hazardous and Noxious Substances (HNS) spill in the marine environment: lethal and sublethal effects of acrylonitrile to the European seabass.

Despite the extensive maritime transportation of Hazardous and Noxious Substances (HNS), there is a current lack of knowledge on the effects posed by HNS spills on the marine biota. Among the HNS identified as priority, acrylonitrile was selected to conduct ecotoxicological assays. We assessed the acute and subletal effects of acrylonitrile in seabass, followed by a recovery phase to simulate the conditions of a spill incident. The work aimed at testing a broad range of biological responses induced by acrylonitrile. Sublethal exposure to the highest two doses increased the fish mortality rate (8.3% and 25% mortality in 0.75 and 2 mg L(-1) acrylonitrile concentrations), whereas no mortality were observed in control and 0.15 mg L(-1) treatments. Additionally, important alterations at sub-individual level were observed. Acrylonitrile significantly induced the activities of Catalase- CAT and Glutathione S-Transferase - GST; and the levels of DNA damage were significantly increased. Conversely, Superoxide Dismutase- SOD - activity was found to be significantly inhibited and no effects were found on Lipid Peroxidation- LPO and ethoxyresorufin O-deethylase - EROD - activity. Following a 7d recovery period, the levels of CAT, GST and EROD fell to levels at or below those in the control. In the 2 mg L(-1) group, SOD remained at the levels found during exposure phase. This study has gathered essential information on the acute and subletal toxicity of acrylonitrile to seabass. It also demonstrated that 7d recovery allowed a return of most endpoints to background levels. These data will be useful to assist relevant bodies in preparedness and response to HNS spills.

Statins: An undesirable class of aquatic contaminants?

Emerging pollutants, such as pharmaceuticals, may pose a considerable environment risk. Hypocholesterolaemic drugs such as statins are among the most prescribed human pharmaceuticals in western European countries. In vertebrates, this therapeutic class disrupts the cholesterol synthesis by inhibiting the enzyme 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR), responsible for the limiting step in the mevalonate pathway. Recently, functional studies have shown that statins competitively inhibit HMGR in vertebrates and arthropods, two taxa that have diverged over 450 million years ago. Importantly, chronic simvastatin exposure disrupts crustacean reproduction and development at environmentally relevant concentrations. Hence, a fundamental question emerges: what is the taxonomic scope of statins-induced HMGR inhibition across metazoans? Here, we address this central question in a large sampling of metazoans using comparative genomics, homology modelling and molecular docking. Sequence alignment of metazoan HMGRs allowed the annotation of highly conserved catalytic, co-factor and substrate binding sites, including residues highjacked for statin binding. Furthermore, molecular docking shows that the catalytic domains of metazoan HMGRs are highly conserved regarding interactions, not only with HMG-CoA, but also with both simvastatin and atorvastatin, the top prescribed statins in Europe and USA. Hence, the data indicates that both statins are expected to competitively inhibit metazoans HMGRs, and therefore all metazoan taxa might be at risk. The environmental relevance of these findings are discussed and research priorities established. We believe that the conceptual framework used in this study can be applied to other emerging pollutants and assist in the design of toxicity testing and risk assessment.

Management of contaminated marine marketable resources after oil and HNS spills in Europe

Different risk evaluation approaches have been used to face oil and hazardous and noxious substances (HNS) spills all over the world. To minimize health risks and mitigate economic losses due to a long term ban on the sale of sea products after a spill, it is essential to preemptively set risk evaluation criteria and standard methodologies based on previous experience and appropriate scientifically sound criteria. Standard methodologies are analyzed and proposed in order to improve the definition of criteria for reintegrating previously contaminated marine marketable resources into the commercialization chain in Europe. The criteria used in former spills for the closing of and lifting of bans on fisheries and harvesting are analyzed. European legislation was identified regarding food sampling, food chemical analysis and maximum levels of contaminants allowed in seafood, which ought to be incorporated in the standard methodologies for the evaluation of the decision criteria defined for oil and HNS spills in Europe. A decision flowchart is proposed that opens the current decision criteria to new material that may be incorporated in the decision process. Decision criteria are discussed and compared among countries and incidents. An a priori definition of risk criteria and an elaboration of action plans are proposed to speed up actions that will lead to prompt final decisions. These decisions, based on the best available scientific data and conducing to lift or ban economic activity, will tend to be better understood and respected by citizens.